A comprehensive review on the neuroprotective potential of resveratrol in ischemic stroke

Stroke is the second leading cause of death and the third leading cause of disability worldwide. Globally, 68 % of all strokes are ischemic, with 32 % being hemorrhagic. Ischemic stroke (IS) poses significant challenges globally, necessitating the development of effective therapeutic strategies. IS is among the deadliest illnesses. Major functions are played by neuroimmunity, inflammation, and oxidative stress in the multiple intricate pathways of IS. Secondary brain damage is specifically caused by the early pro-inflammatory activity that follows cerebral ischemia, which is brought on by excessive activation of local microglia and the infiltration of circulating monocytes and macrophages. Resveratrol, a natural polyphenol found in grapes and berries, has shown promise as a neuroprotective agent in IS. This review offers a comprehensive overview of resveratrol's neuroprotective role in IS, focusing on its mechanisms of action and therapeutic potential. Resveratrol exerts neuroprotective effects by activating nuclear factor erythroid 2-related factor 2 (NRF2) and sirtuin 1 (SIRT1) pathways. SIRT1 activation by resveratrol triggers the deacetylation and activation of downstream targets like peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and forkhead box protein O (FOXO), regulating mitochondrial biogenesis, antioxidant defense, and cellular stress response. Consequently, resveratrol promotes cellular survival and inhibits apoptosis in IS. Moreover, resveratrol activates the NRF2 pathway, a key mediator of the cellular antioxidant response. Activation of NRF2 through resveratrol enhances the expression of antioxidant enzymes, like heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1), which neutralize reactive oxygen species and mitigate oxidative stress in the ischemic brain. Combined, the activation of SIRT1 and NRF2 pathways contributes to resveratrol's neuroprotective effects by reducing oxidative stress, inflammation, and apoptosis in IS. Preclinical studies demonstrate that resveratrol improves functional outcomes, reduces infarct size, regulates cerebral blood flow and preserves neuronal integrity. Gaining a comprehensive understanding of these mechanisms holds promise for the development of targeted therapeutic interventions aimed at promoting neuronal survival and facilitating functional recovery in IS patients and to aid future studies in this matter.


Introduction
Stroke is the second largest reason for mortality and also the third greatest factor of disabilities worldwide [1].Depending on the underlying pathology, strokes can be classified as either ischemic or hemorrhagic; worldwide, 32 % are hemorrhagic and 68 % of strokes are ischemic [1,2].
Ischemic stroke (IS) is caused by a disruption of blood flow to the brain.In IS, two different damage regions have been determined: the lesion core, where cells die quickly, and the penumbra (the area around the lesion core), where cells are functionally compromised but may recover and restore function [3][4][5].IS is the world's second biggest cause of mortality, with 5.9 million deaths and 102 million disability-adjusted life years lost [5].IS starts a series of excitotoxicity processes, like ATP depletion, ionic dysregulation, inflammatory processes, oxidative injury, apoptosis, angiogenesis, raised glutamate release, excessive generation of free radicals, apoptosis, and necrosis; all these occurrences ultimately lead to cell death [2,6,7].
Tissue plasminogen activator (tPA) is currently the only FDA-approved treatment for IS [8].This treatment option comes with a series of limitations such as a narrow therapeutic time window, meaning that not every patient is eligible for treatment [9].Even in those who are eligible and receive tPA therapy, outcomes depend on the severity of the stroke and may not be significantly improved.This may be due to the reperfusion injury caused by restoring blood flow to the ischemic area which initiates an inflammatory response, generation of free radicals, increased activity in lipases, and endonucleases and therefore worsening of neurological status [10].These limitations and failure to recover the dying neurons urge us to search for an alternative approach or additive treatments to combine with tPA.
Resveratrol and its anti-inflammatory, anti-apoptotic, and anti-oxidant effects have shown significant health benefits in multiple diseases such as cancer, cardiovascular disease, diabetes and infectious disease [11].
Resveratrol (3,5,4′-trihydroxystilbene) (PubChem CID: 445154) is a naturally occurring phytosterol that resembles estrogen and is mostly present in grapes, peanuts, blueberries, red wine, and other dietary components [12].Multiple studies have demonstrated that resveratrol presented protective effects in IS, it can mediate blood pressure and lipid profiles which are the main key factors in managing and preventing stroke [13][14][15][16][17].In this review, we aim to summarize how resveratrol may have a beneficial impact on stroke outcomes and to discuss its therapeutic potential based on previous published literature and laboratory findings.

Ischemic stroke cascade
When the brain is deprived of the proper blood supply, the ischemic core undergoes infarction and neuronal death immediately.Following the acute phase, multiple processes occur around the ischemic core called the brain penumbra leading to secondary brain injury [18].
As the brain undergoes ischemia, energy stores drop, causing an ionic imbalance and the release of excitotoxic neurotransmitters such as glutamate.The increased release of glutamate and inhibition of its reuptake lead to the overactivation of ionotropic N-Methyl-D-aspartate (NMDA) and α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.Glutamate receptor activation promotes fatal amounts of calcium to enter the cell.This calcium overload leads to the activation of proteases, phospholipases and nucleases which further disrupt membranes and proteins essential for cell function and generate free radicals that are damaging to neurons [19][20][21].These events initiate subsequent damage caused by oxidative stress, inflammation, and rupture of the blood brain barrier (BBB) [22,23].
Injured neurons release damage-associated molecular patterns (DAMP) which are responsible for triggering postischemic inflammation [24,25].DAMPs like interleukin (IL)-33, heat shock proteins (HSP) and adenosine are involved in IS [25,26].Immune cells detect these danger signals and activate intracellular signaling pathways essential for triggering immune responses [27].The brain's resident inflammatory cells, microglia, generate pro-inflammatory cytokines like IL-1β, tumor necrosis factor (TNF)-α, and IL-6 in the early phases of IS [28].The proinflammatory transcription factor kappa B (NF-kB) performs a critical role in cytokine production and the inflammatory response [29].These cytokines cause endothelial damage, resulting in increased BBB permeability [30].The microvascular damage and BBB breakdown exacerbate the inflammation in the ischemic region by increasing the permeability and migration of peripheral blood cells to the brain [30,31].This innate immune system further provokes BBB dysfunction by releasing even more cytokines, reactive oxygen species (ROS), and the chemokine matrix metalloproteinase (MMP)-9, creating cerebral edema and expanding the infarct region [32,33].These processes, which are all comprised of inflammation, oxidation, autophagy, necrosis, and apoptosis lead to neuronal death [34].Mediating the immune response has shown neuroprotective effects against IS and therefore improving stroke outcomes [35].

Role of microglia in ischemic stroke
IS is complicated and involves several stages,including BBB disruption, oxidative stress, neuroinflammation, neuroexcitotoxicity, and microglial activation [36].After ischemia, many factors such as necrotic cells, ROS, and damaged tissues cause inflammation, which in turn triggers the activation of inflammatory cells such as microglia [37].The CNS's resident macrophages, known as microglia, which make up about 20 % of the overall glial population, are the first cells to respond to IS [38].Although microglia activation is deleterious in IS, it is essential for boosting neurogenesis, reducing neuronal death, and enhancing functional recovery following cerebral ischemia [36].Neuroinflammation is criticalin the development and progression of ischemic stroke [39].Microglial-mediated neuroinflammation is not an independent process and has intricate interactions with other pathological mechanisms like oxidative/nitrative stress, excitotoxicity, necrosis, apoptosis, pyroptosis, autophagy, and adaptive immune reactions [40].
Microglia, when activated, variously express numerous channel proteins, receptors, and enzymes that are associated with boosting or preventing inflammatory processes, resulting in them being an area of intervention for ischemic stroke [40].
Microglia are classified into three kinds: M0 (surveillance), M1 (pro-inflammatory), and M2.(anti-inflammatory) [7].M0 is mainly accountable for surveillance and exhibits poor phagocytosis and inactivity [41,42].M1 phenotypic polarization results in the production of pro-inflammatory compounds that impair CNS recovery, while M2 phenotypic polarization results in the release of cytokines that are anti-inflammatory and promote tissue repair and regeneration [43].M1 microglia release pro-inflammatory molecules like IL-1, IL-6, IL-1β, TNF-α, and IFN-γ which have cytotoxic effects on neurons by increasing the production of inducible nitric oxide synthase (NOs), which leads to the death of neurons [36,44].These cytokines andMMPs exert essential functions in BBB destruction in IS.They create a rise in adhesion molecules and inflammatory blood cells, particularly neutrophils, which penetrate via the compromised BBB [45].Compared to M1 microglia, M2 microglia have a greater ability to initiate phagocytosis of dying cells, which can limit the ensuing inflammation and facilitate tissue regeneration [46,47].

Role of astrocytes in ischemic stroke
Astrocytes are the most prevalent kind of glial cell in the brain, comprising approximately 40 % of all brain cells.Astrocytes are classified into three distinct groups based on their shape and spatial organisation:, protoplasmic astrocytes in the grey matter, radial astrocytes that surround the ventricle, and fibrous astrocytes located in the white matter [48].Astrocytes become active quickly and display two distinct functional phenotypes: the neurotoxic type A1 astrocytes, mostly triggered by inflammation and the neuroprotective A2 type reactive astrocytes, triggered by ischemia.
Astrocytes undergo major alterations in their shape, activity, and molecular expression profile during an IS [49].Astrocytes become activated during minutes of brain ischemia due to cytokines generated by injured neurons and glial cells in the penumbra and core of the infarct.This is also known as reactive astrogliosis, and it is marked by cell proliferation, hypertrophy, and elevated expression of the glial fibrillary acidic protein (GFAP).It also alters the expression of several molecules that affect cell structure, intracellular signal transmission, gene transcription, energy metabolism, and membrane transport proteins.In addition to supporting structure and metabolism, they can protect the BBB, control blood vessel tone in reply to neuronal activity, remove excess neurotransmitters (glutamate homeostasis), balance oxidative stress, and encourage the development and maintenance of synapses [50][51][52][53].Astrocytes are involved in a variety of ischemia signal-induced procedures, like excitotoxicity, oxidative stress, metabolic dysregulation, edema production, scar-border development, neuroinflammation and finally apoptosis and necrosis of neurons [53].Astrocytes may be activated by inflammatory substances generated by microglia, including transforming growth factor-alpha (TGF-α), IL-6, leukemia inhibitory factor (LIF), and TNF-α.In addition, dying neurons and endothelial cells contribute to astrocyte activation.They mainly generate cytokines to control the activation and proliferation of astrocytes [48].Numerous inflammatory compounds, including TNF-α, IL-1α, and IFN-γ, along with free radicals, like NO, peroxynitrite, and superoxide dismutase are generated and released via reactive astrocytes (RA).These agents either indirectly or directly result in neuroinflammation, which in turn causes neuronal apoptosis and necrotic death.Moreover, RAs release cytokines that prevent inflammation from happening [54][55][56].The NF-κB signalling pathway promotes the formation of inflammatory-neuronal astrocytes.These astrocytes cause neuronal apoptosis through reregulating complement cascade genes, releasing inflammatory cytokines, and decreasing the production of SPARCL1, GPCG4/6, and ThBS1/2 with neurotrophic operation [57][58][59].The JAK2/STAT3 signalling pathway-mediated neuroprotective kind astrocytes up-regulate several neurotrophic factors and support neuronal survival and development, indicating that this kind of astrocyte might serve a beneficialrepair role [60].

Resveratrol: bioavailability and therapeutic effects
Resveratrol (3,5,4′-trihydroxystilbene) (PubChem CID: 445154) is a naturally occurring phytosterol that resembles estrogen and is mostly present in grapes, peanuts, blueberries, red wine, and other dietary components [12].Resveratrol has a quick rate of absorption and is absorbed in substantial quantities by enterocytes [61,62].However, plasma levels of resveratrol are often low owing to high levels of intestine and liver metabolism [62].When resveratrol reaches the gastrointestinal system, it undergoes quick and substantial biotransformation before being distributed to different organs, where it becomes accessible and active [63].Absorbed resveratrol in intestinal enterocyte cells undergoes sulfation and glucuronidation processes [64].Resveratrol that has been conjugated leaves the cell through the transporters in the apical and basolateral membranes, while a tiny amount of resveratrol that has not been conjugated leaves the enterocyte through the basolateral membrane [63].Resveratrol and conjugated metabolites pass through the small intestine's apical membrane and into the large intestine.Here they are metabolized by gut bacteria to produce dihydroxytrans-stilbene, lunularin and dihydroresveratrol [65].Resveratrol and metabolites that leave the enterocyte enter the portal circulation and the liver, and thus undergo further conjugation [66].Furthermore, conjugated resveratrol and metabolites pass through enterohepatic circulation, exiting the liver to be reabsorbed in the intestine following hydrolysis and then reaching the portal circulation and returning to the liver for additional metabolism [64].Resveratrol and its metabolites reach the systemic circulation via the liver and are transported by binding to blood proteins like lipoproteins, hemoglobin, and albumin [67].The kidneys are involved in the metabolism of resveratrol as well, which results in the excretion of polarised resveratrol metabolites [64].Resveratrol not only has a quick metabolism, but it also is excreted quickly.A study on rats has shown that 49-60 % of the resveratrol is eliminated in the urine, from the remaining 77-80 % being absorbed in the intestine [61].Thus, 75 % of the total amount of resveratrol that is ingested is excreted [68].
The residual quantity of resveratrol undergoes metabolism, with the maximum reported concentration of free resveratrol being 1.7-1.9% [69].The levels of resveratrol peak 60 min following ingestion.Another study found that within 6 h, there was a further rise in resveratrol levels.This increase can be attributed to intestinal recirculation of metabolites.In the intestines, these metabolites are reabsorbed after being hydrolyzed into their free form [69].
Resveratrol (3,4,5 -trihydroxytrans-stilbene) is a polyphenolic molecule from the stilbene class that occurs in two isomers: cis and trans [70].Stilbenes, a type of secondary metabolite, eliminates free radicals and protects against chronic illnesses like diabetes, cancer, heart disease, and arteriosclerosis [71].They also help with aging [72].However, resveratrol may provide additional health advantages, since various studies have shown that it has antioxidant, anti-inflammatory, neuroprotective, and chemotherapy-protective properties [73].
Resveratrol is commonly found in red grapes (the richest source), cocoa, peanuts, wine, grape juice, berries of Vaccinium species like blueberries, cranberries and bilberries [74].It was found that resveratrol can lower blood pressure in hypertensive rats, providing a novel treatment option for cardiovascular disease [14].This substance modulates cellular immunity [11].In addition, resveratrol may protect against neurological illnesses including Parkinson's and Alzheimer's [75].Recently, researchers studied the therapeutic potential of resveratrol in hemostatic diseases related with COVID-19.This compound's anti-clotting and anti-inflammatory activities have been proven to have a crucial impact in lowering COVID-19 mortality [76].It is also helpful in various areas, including mitochondrial malfunction, oxidative stress, angiogenesis, apoptosis and inflammation.It inhibits platelet aggregation and has cardioprotective effects [77].

Effect of resveratrol on astrocytes and microglia
Resveratrol treatment has shown to improve neuronal dysfunction, infarct volume, and neuronal morphological alterations in MCAO animals.Meanwhile, pro-inflammatory microglia activation and inflammatory factor productions were suppressed.CD147 and MMP-9 levels were elevated in primary microglia.Resveratrol inhibited the CD147/MMP-9 axis in OGD/R microglia.All leukocytes, platelets, and endothelial cells contain the transmembrane glycoprotein CD147, which has been demonstrated to be a key mediator of inflammation and immune responses [78].
Activated Smo can enhance neurological performance by controlling oxidative stress, inflammation, apoptosis, neurogenesis, oligodendrogenesis, and axonal remodeling.More investigations have shown that resveratrol can activate Smo.However, it is still unclear whether resveratrol suppresses the activity of microglial cells with Smo.Studies have found that the Smo receptor could be a therapeutic target of resveratrol, assisting to decrease microglial activity in the acute phase of stroke [79].Resveratrol has a neuroprotective impact versus IS, which is partly due to its stimulation of JAK2/STAT3 and PI3K/AKT/mTOR axis.Resveratrol may indirectly stimulate the PI3K/AKT/mTOR axis via stimulating JAK2/STAT3 [80].
The expressions of iNOS and NF-κB p65 subunits in microglial cells were elevated after a 24-h exposure to LPS/IFNγ, and the releases of TNFα and IL-1β were also enhanced.Resveratrol decreased the expressions of iNOS and NF-κB p65 subunits as well as the releases of proinflammatory cytokines [82].
Resveratrol also increases AMPK and inhibits GSK-3β (glycogen synthase kinase 3 beta) activity in astrocytes, which release energy, makes ATP available to neurons and reduces ROS. .Furthermore, oligodendrocyte survival is boosted by resveratrol, which may help to preserve brain homeostasis following a stroke [81].Based on these findings, resveratrol may be regarded as a novel therapeutic option for improving the symptoms of stroke.It can influence neuronal function and also significantly lower neurotoxicity by changing glial function and signaling.

Neuroprotective roles of resveratrol
Resveratrol has neuroprotective benefits in both IS and intracerebral hemorrhage.Atherosclerosis is regarded as a risk factor for IS.In this regard, resveratrol may suppress platelet activation and aggregation caused by collagen, adenosine diphosphate, and thrombin.The postulated mechanism encompasses the suppression of tissue factor gene expression or the manufacture of prothrombotic agents [83].Intracranial hypertension and cerebral edema are frequent consequences of a cerebral infarction and can lead to death.A variety of therapies that effectively combat cerebral edema have been created in animal experiments, a number of which have been evaluated in clinical trials; among them, resveratrol has shown edema reducing effects.Although resveratrol is a highly hydrophobic molecule, it is exceedingly difficult to penetrate a membrane like the BBB.However, an alternate administration is through the nasal cavity in the olfactory area, which results in a more pleasant route for the patient.Resveratrol has multiple approaches of action associated to its effects on stroke, since the molecule interacts with a broad range of enzymes and receptors, enhancing stress resistance and lowering apoptosis [84][85][86].In addition to apoptosis, several other pathophysiologic processes like inflammation, oxidative stress, and ionic imbalance also work in concert to cause brain damage and neuronal death following a stroke [87].Resveratrol is being widely investigated as a potential therapeutic agent in IS due to its anti-inflammatory, antioxidant, anti-tumorigenic,anti-aggregation, and edema reducing properties [88] (Fig. 1).

Anti-inflammation
Resveratrol's anti-inflammatory effects have been demonstrated in many studies.One of the major roles of resveratrol in attenuating inflammation is through the activation of SIRT1 [89,90].Resveratrol is known as the most effective up regulator of SIRT1 [91].SIRT1 is a deacetylase that plays a key role in maintaining immune tolerance and regulating T cell function [92,93].Evidence-based studies have implicated the effects of SIRT1 activation in reducing inflammation and alleviating immune response [94,95].SIRT1 is widely expressed in the CNS and therefore, overactivation of SIRT1 can lead to regulating postischemic neuroinflammation [96].One of resveratrol's substrates is p65/RelA which is a member of the NF-kB transcription factor that significantly increases cytokine release and leukocyte activation [97,98].Cytokines released due to ischemic conditions disrupt the BBB and increase its permeability allowing monocyte, neutrophil, and leukocyte infiltration which aggravates inflammation in the ischemic area [28].Resveratrol binds to SIRT1 and modulates its structure, facilitating its binding to substrates [99].This enhances RelA acetylation which results in inhibition of the NF-kB dependent cytokine production like, IL-1β, IL-6, and MMP-9 [100,101].Hence, resveratrol exhibits the potential to protect the BBB against leakage and immune cell infiltration and therefore, limit neuron loss [102].A study performed on cortical mixed glial cells exposed to hypoxia/hypoglycemia demonstrated that IL-6 gene expression and excretion was reduced following resveratrol treatment in a dose dependent manner [103].In a mouse model of MCAO, resveratrol administration exhibited dose-dependent reductions in IL-1β, IL-6, TNF-α, and ROS production in the ischemic cortex, and ultimately decreased infarct volumes as compared to the control group [104,105].Shin et al. evaluated IL-1β and TNF-ɑ mRNA and protein levels in an MCAO model.They found that the mRNA and protein levels of these two cytokines were markedly diminished in the resveratrol-treated group [105].Another study demonstrated that myeloperoxidase activity, which indicates neutrophil infiltration, was reduced in MCAO rats treated with resveratrol [106].Resveratrol was found to lower cytokine levels in both the central nervous system and the peripheral blood, suggesting that this treatment option may help preserve the integrity and structure of the blood-brain barrier [107].Resveratrol can also suppress the CD147/MMP-9 pathway, which in turn inhibits the production of cytokines from microglia in the ischemic brain, according to a recent animal research [78].IS etiology appears to be significantly influenced by CD147 and MMP-9 [108].The experiment's findings demonstrated that 24 h following brain ischemia, MPO activity was noticeably increased.However, this increase shown a substantial Fig. 1.Neuroprotective role of resveratrol in IS.Following cerebral ischemia, the secondary damage continues to affect the brain and injure neurons due to immune cell infiltration, oxidative stress and BBB disruption.Resveratrol holds the potential to inhibit inflammatory cytokine release and preserve the BBB integrity through the activation of the SIRT1 and Nrf2 pathway.Resveratrol upregulates the expression of anti-oxidant genes including Superoxide dismutase (SOD)2 and NAD(P)H quinone oxidoreductase 1 (NQO-1).Resveratrol also stimulates the expression of the Bcl-2 anti-apoptotic gene while suppressing the expression of apoptotic genes like Bax, caspase and Hypoxia-inducible factor 1-alpha (HIF-1a).Resveratrol is capable of reducing cerebral edema by preserving the BBB integrity and reducing the expression of aquaporin (AQP)-4.The SIRT1 pathway leads to increased nitric oxide (NO) production.Increased NO along with the reduction in angiotensin II and endothelin 1 contribute to resveratrol effects on regulating blood pressure and cerebral blood flow.decrease following resveratrol medication [109].Resveratrol dramatically lowered the amounts of cerebral infarcts, neuronal damage, MPO activity, and evans blue (EB) content in addition to neurological impairment scores.TLR4, NF-κB p65, COX-2, MMP-9, TNF-α, and IL-1β all had greater levels of expression after cerebral ischemia, whereas resveratrol decreased these amounts [109].

Anti-oxidative
Oxidative stress is when cells are exposed to molecular oxygen or its derivatives, particularly ROS, and the cells fail to protect themselves against cellular damage [110].ROS production and oxidative stress, damage neurons in ischemic situations and play a key role in ischemic reperfusion injury [111].Resveratrol applies its anti-oxidant effects through diverse mechanisms and pathways that activate anti-oxidant enzymes [112].
One of the major pathways that resveratrol activates to fight against oxidative stress is the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway.The Nrf2 transcription factor is responsible for anti-oxidant gene regulation and protects cells from oxidative damage [113].During normal conditions, Nrf2 stays in its inactive form by binding to the keap1 protein [114].During stressful situations and ROS accumulation, the keap1 protein dissociates from Nrf2 and leads to anti-oxidant gene expression [115,116].Resveratrol can lead to the dissociation of keap1 from Nrf2 and increase its active form [117].When Nrf2 is dissociated from keap1, it translocate into the nucleus and heterodimerizes with small Maf proteins, which then bind to the cell's DNA and lead to the transcription of Nrf2 target genes including heme oxygenase-1 (HO-1) [118].HO-1 can reduce the overproduction of inflammatory cytokines (e.g., TNF-ɑ, IL-6) by inhibiting the NF-kB signaling pathway [119].The enzyme HO-1 breaks down free heme to produce carbon monoxide (CO), free iron, and biliverdin, which is quickly converted to bilirubin via biliverdin reductase [120].Unconjugated bilirubin, biliverdin, and CO block NADPH oxidase complexes, which are significant contributors to excitotoxicity [121][122][123].HO-1 is a Nrf2-regulated gene with significant antioxidant, anti-inflammatory, antiapoptotic, and antiproliferative properties [124].Once NADPH oxidase is hyperactive, the ensuing oxidant generation stimulates Nrf2 and thus promotes HO-1, which produces bilirubin, this inhibits NADPH oxidase activity via feedback.Therefore, HO-1 regulates cellular oxidative stress.[125].Furthermore, when iron free ions are exposed to oxidants like superoxide and hydrogen peroxide, they can form very reactive and deadly oxidant hydroxyl radicals, which can cause neurotoxicity [126].In addition to biliverdin and bilirubin, CO generated by HO-1 induction inhibits the NADPH oxidase [127].Low amounts of CO can stimulate the enzyme guanylate cyclase, mimicking the physiological action of NO.The product of this enzyme's activity, cyclic GMP (cGMP), has the ability to activate protein kinase G (PKG), that in turn causes neurotropic activity in neurons through AKT kinase activation.Therefore, therapies which raise cGMP concentrations in neurons may be effective in counteracting excitotoxicity [127].Since NO also aids in the generation of peroxynitrite, enhancing neuronal generation of NO isn't a suitable way to achieve this.
The expression levels of HO-1 in ischemic cerebrovascular disease patients are significantly increased and positively correlated with the severity of the disease [128][129][130][131]. Post-ischemic upregulation of HO-1 appears to be part of the response mechanisms of the brain to reduce neuronal damage [128].
Findings from previous studies suggest that Nrf2 activation can significantly reduce brain injury following IS and lead to better outcomes [132].A study on the primary Culture of Rat Cortical Neurons indicated that resveratrol significantly reduces brain injury induced via oxygen/glucose deprivation/reoxygenation (OGD/R) in the rat cerebral cortex by upregulating Nrf2 [133].They used Western blot analysis to show that levels of anti-oxidant molecules such as superoxide dismutase 2 (SOD2) protein and NAD(P)H quinone oxidoreductase-1 (NQO-1) were increased in rat cultures treated with resveratrol compared to the normal group.
Abdel-Aleem, Ghada A. et al. demonstrated that resveratrol can protect the brain against ischemia-reperfusion injury by regulating the DJ-1 protein [134].This protein has a key role in anti-oxidation and cell survival, but its function is disrupted during ischemia-reperfusion injury and fails to protect neurons [117].They found that resveratrol reduced oxidized forms of DJ-1 levels, resulting in increased survival in rats with brain damage.
Malondialdehyde (MDA) is a commonly used biomarker for detecting oxidative stress in cells undergoing injury [142].MDA can react with cell structures such as phospholipids and nucleic acids, which can lead to immune system dysfunction [143].Studies have shown that serum MDA levels are increased in IS patients [144,145].Resveratrol administration has been shown to reduce MDA levels and lipid peroxidation in the mouse brain [146].

Anti-apoptosis
Apoptosis, programmed cell death, plays a crucial role in various pathological processes, such as stroke [147].It may take several hours or days for apoptosis to take place in the brain penumbra following an IS [148].Due to ionic imbalance and calcium overload in IS, neurons may undergo apoptosis, which leads to a significantly greater amount of cell death [149].Resveratrol has shown pro-apoptotic properties in cancer [150] while it possesses an anti-apoptotic property in acute CNS insults [138].Resveratrol induces its anti-apoptotic effects through multiple pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway and the extracellular signal-regulated kinase (ERK) pathway.Activation of these pathways can promote cell survival and inhibit apoptotic signaling [80].Resveratrol holds the potential to upregulate the Bcl-2 family proteins, which play a key role in apoptosis suppression while downregulating the expression of pro-apoptotic proteins such as Bax [151].Caspases are key mediators of apoptosis.Resveratrol has been shown to inhibit caspase activation, particularly caspase 3, which is responsible for carrying out the apoptosis process and therefore leading to cell survival [152].Resveratrol can prevent mitochondrial membrane depolarization, preserve adenosine triphosphate (ATP) production, and inhibit the release of cytochrome c.In addition, mitochondrial lipid peroxidation (LPO), protein carbonyl, and intracellular hydrogen peroxide (H2O2) content were significantly reduced in the resveratrol treatment group, while the expression of HSP70 and metallothionein were restored [153].Metallothionein and HSP70 are stress proteins involved in protection against oxidative damage [154].Oxidative damage following a stroke can induce apoptosis in cells [155].Resveratrol's anti oxidative effects, discussed earlier, also contribute to reducing the risk of apoptosis.Wang et al. demonstrated that resveratrol reduced neurocyte apoptosis, as evidenced by an enhanced Bcl-2/Bax ratio, and lowered the levels of apoptotic cells (TUNEL positive cells) [156].In female rats treated with resveratrol for acute global cerebral infarction, Bcl-2 levels were significantly elevated, but p53 levels as an apoptotic marker were significantly decreased [157].

Edema reducing effects
Cerebral edema can aggravate stroke outcomes.This lethal condition occurs within 48 h post IS and peaks 3-5 days after stroke occurrence [158].Ischemic conditions cause tissue necrosis and basal membrane breakdown, leading to BBB impairment [159,160].This allows serum proteins to improperly shift from the blood to the brain and lead to a type of vasogenic edema which significantly affects the degree of neurologic deficit [158].A study performed on rats demonstrated that resveratrol inhibited brain edema in IS.Resveratrol also decreased the levels of plasma membrane channels responsible for water hemostasis called Aquaporin (AQP)-4 [161].Resveratrol's edema reducing effects are due to its impacts on preserving BBB integrity and its ability to lower AQP-4 levels.

Effects of resveratrol on blood pressure and cerebral blood flow
The mechanisms underlying the blood pressure-lowering effects of resveratrol are not fully understood but may involve multiple pathways.Resveratrol has been found to enhance NO production, a potent vasodilator, which can lead to relaxation and widening of blood vessels.By promoting NO-mediated vasodilation, resveratrol may help improve cerebral blood flow (CBF) [162].In addition, resveratrol has been shown to inhibit angiotensin II, a hormone that can constrict blood vessels and lead to high blood pressure [163] The ability of resveratrol to modulate these pathways may contribute to its beneficial effects on blood pressure regulation.Moreover, resveratrol has been reported to possess antioxidant and anti-inflammatory properties as discussed earlier, which could also play a role in blood pressure regulation.Oxidative stress and chronic inflammation are known to contribute to endothelial dysfunction and vascular damage, both of which are associated with hypertension [13,164].By reducing oxidative stress and inflammation, resveratrol may help improve endothelial function and maintain healthy blood pressure levels and therefore, improve CBF [165,166].It has been suggested that resveratrol can influence the release and activity of vasoactive substances, such as endothelin-1 and prostaglandins, which can modulate the diameter of blood vessels in the brain [167,168].By modulating these vasoactive substances, resveratrol may regulate cerebral microcirculation and impact CBF.

Animal experiments with resveratrol in IS
Multiple studies have experimented with the effects of resveratrol on various stroke outcomes.Shin et al. showed that 5 mg/kg intravenous (IV) resveratrol reduced infarction volume by 36 % in an MCAO mouse model.The neuroprotective effects are due to inflammatory suppression as well as ROS inhibition in the ischemic cortex [105].Another study by the same authors confirmed their previous findings when resveratrol reduced total infarct volume by 45 % [91].Their results also showed that resveratrol activates the transcription factor cAMP-response-element binding protein (CREB).CREB enhances cortical circuit plasticity and is involved in forming new connections which lead to recovery from stroke motor deficits [169].Rats pre-treated with nanostructured lipid carriers containing resveratrol (NR) for 10 days before MCAO showed improved behavioral tests and decreased infarct volume in a dose-dependent manner.The increased activities of caspases 3 and 9 as well as cytokines (IL-1, 6, and TNF-α) in the MCAO group were considerably prevented by 500 μg of NR administration.This study indicates that resveratrol holds the potential to improve stroke outcomes before ischemia as a pre-treatment strategy [104].Resveratrol treatment after a single or repeated mild stroke has been shown to decrease brain injury, prevent cerebral edema, protect endothelial cells and preserve BBB function [170].Faggi et al. found that resveratrol treatment significantly reduced the infarct volume in mice with MCAO.They did minimize the infarct volume when administered at higher doses (resveratrol 6.8 mg/kg).According to the findings of this study, resveratrol may be an effective ready-to-use treatment for treating post-ischemic brain injury [171].Another study found that trans-resveratrol treatment significantly reduced infarct volume and prevented motor impairment, increased glutathione, and decreased MDA levels compared to the control group, suggesting that trans-resveratrol could be one of the medications used for minimizing neurologic deficits due to stroke [172].In the delayed phase after stroke, resveratrol treatment via gavage has been shown to be useful for reducing infarct volume and improving neurological impairments.The increased MMP-2 and vascular endothelial growth factor levels may play a role in the neuroprotective effect of resveratrol treatment by causing angiogenesis [16].Another research article emphasized the importance of resveratrol in the treatment of ischemic stroke due to its capacity to preserve the structure and function of ischemic neurovascular units.Resveratrol significantly reduced neurological deficits as evaluated by several scoring techniques, brain infarct volume, and brain edema [153].The TRPC6-MEK-CREB and TRPC6-CaMKIV-CREB pathways were identified by Lin et al. as potential mechanisms by which resveratrol protects neurons from ischemia injury.After cerebral ischemia/reperfusion injury, pretreatment with resveratrol for 7 days significantly reduced neurological deficits, and the infarct volumes were associated with increased TRPC6 and p-CREB activity [173].
Resveratrol treatment significantly decreased infarct sizes, improved neurobehavioral deficits, and prevented neuronal cell death in both in vivo and in vitro models of recurrent ischemic stroke.Resveratrol treatment significantly enhanced the intracellular NAD + /NADH ratio, as well as AMPK and SIRT1 activities and, decreased energy ATP requirements during ischemia, consequently suggesting the neuroprotective properties of resveratrol [174].Fang et al. found that resveratrol was able to decrease the extent of cerebral infarction, brain water content, neuronal apoptosis, myeloperoxidase levels (which are expressed in immune cells), and cerebral TNF-α production in a rat model of focal cerebral ischemic reperfusion injury [175].Pretreatment with 50 mg/kg resveratrol for 7 days was shown to protect against cerebral ischemia/reperfusion injury via enhanced anti-apoptosis, anti-inflammation, and autophagy activation.Resveratrol significantly lowered the neurologic deficit, cerebral infarct volume, the levels of caspase-3 and IL-1β, but also significantly increased the ratios of Bcl-2/Bax [176].When given 6 days before MCAO, resveratrol significantly decreased neurological deficits and infarct volume.The authors hypothesized that resveratrol's neuroprotective effects may be via inhibiting phosphodiesterase, and subsequently activating the AMPK/SIRT1 signaling pathway, and lowering ATP energy  consumption in neurons during ischemia [177].Intra-carotid artery administration of resveratrol polymeric nanoparticles showed significant protection against cerebral ischemia/reperfusion injuries, as evidenced by improved neurological functions, decreased infarction volumes, preservation of the BBB, prevention of brain edema, attenuation of oxidative stress, inhibition of neuronal apoptosis, and promotion of neurogenesis through increased expression of brain-derived neurotrophic factor and Bcl-2/Bax ratio [178].The brain-derived neurotrophic factor is involved in increasing neuroplasticity and stroke recovery.Treatment with resveratrol after MCAO improved neurobehavioral deficit, brain water content, infraction, and cerebral cortex histological alterations.In addition, resveratrol prevented the declines in phospho-Akt and phospho-GSK-3 protein levels that were induced by MCAO injury [179].Different neurological scoring tests, including cylinder test, spontaneous motility, righting reflex, horizontal bar test, forelimb flexion, actophotometer, rotarod, Randall Sellito and Von Frey were all improved by chronic doses of resveratrol (20 mg/kg) for 21 days.Resveratrol also decreased the immobility time forced swim test and the Morris water maze memory deficit [180].When resveratrol was administered during both gestation and lactation (2 weeks prior to the hypoxic-ischemic in pups), the greatest recoveries were observed.The authors hypothesize that resveratrol affects brain metabolism, especially the astrocyte-neuron lactate shuttle, which contributes to neuroprotective properties [181].Pretreatment with resveratrol (20 or 40 mg/kg) significantly lowered the cerebral edema, infarct volume, lipid peroxidation products, and inflammatory markers, including IL-1β, IL-6, TNF-α, NF-κB p65 subunit and significantly increased the antioxidant capacity by enhancing the activities of glutathione peroxidase, catalase, SOD; and signified an increase in HO-1, and Nrf2 [182].Intraperitoneal administration of resveratrol at a dose of 50 mg/kg reduced cerebral ischemia reperfusion damage, brain edema, and BBB malfunction [183].Outcomes of resveratrol on IS from in vivo studies are summarized in Table 1.

In vitro studies with resveratrol and stroke
Oxygen glucose deprivation (OGD) is a frequently used technique to mimic stroke and investigate molecular mechanisms underlying the condition.The OGD model is widely used to evaluate the possible therapeutic options for stroke.Pretreatment with resveratrol was able to significantly lower astrocytic activation after OGD/reperfusion in vitro [195].Resveratrol showed beneficial impacts in treating neuronal damage caused by OGD.It up-regulated PPAR-α expression in cultured neurons under OGD conditions, as well as suppressed of MMP-9 mRNA expression [214].Resveratrol exerts anti-apoptotic actions in OGD, and it contributes to ERK by inhibiting MMP-9 production [215].ERK plays a key role in almost every cell function and regulates antiproliferative events, including apoptosis [216].Faggi et al. demonstrated that valproate at 1 nmol/mL generated synergistic neuroprotection with resveratrol (3 nmol/mL) in primary neurons exposed to OGD [171].Narayanan et al. concluded that loss of Nrf2 decreased resveratrol potential for neuroprotection and that this pathway plays a crucial role in resveratrol neuroprotective effects [217].Another in vitro model showed that resveratrol inhibits neuronal apoptosis after OGD/reperfusion and boosts Nrf2 activation in a dose dependent manner.Resveratrol administration at different times (pre and post stroke) led to different outcomes (Table 2) [133].Another study revealed that resveratrol treatment decreased TUNEL positive cells while increasing cell viability through the activation of the sonic hedgehog (Shh) pathway [218].Another in vitro model found that cell viability, SIRT1 activity, AMPK activity, NAD + /NADH ratio, and ATP levels were improved with resveratrol [219].Resveratrol treatment 24 h prior to OGD/reperfusion was able to increase cell viability, decrease cell proliferation and reduce inflammatory cytokines.Expression levels of the Shh proteins (Smo, Ptc-1, and Gli-1) were significantly increased.Resveratrol was also able to inhibit the expression of GFAP, S100β, and Vimentin proteins.These proteins are markers of stress and CNS insult [220,221].Table 2 summarizes the effects of resveratrol in vitro experiments.

Clinical trials of resveratrol in ischemic stroke
Few clinical trials that measure the effectiveness of resveratrol on stroke patients.A randomized double-blinded trial assessed the effects of resveratrol on IS patients [228].Three resveratrol capsules (each containing 170 mg) were given to patients 24h after stroke and were continued for 30 days.Systolic and diastolic blood pressures and the National Institute of Health Stroke Scale (NIHSS) were evaluated at the stroke onset and after discharge.Also, the Barthel index and Modified Rankin Scale (MRS) were performed 3 months following resveratrol consumption.They found that resveratrol had no effects on any of the factors mentioned in IS patients compared to the placebo group.Another study measured the effects of resveratrol in combination with r-tPA in IS.Patients were divided into two groups, r-tPA + placebo and r-tPA + 2.5 mg/kg resveratrol (maximum 250 mg).They concluded that resveratrol could extend the narrow therapeutic time window of r-tPA.They found that r-tPA + resveratrol inhibits the effects of MMP-2 and MMP-9 and improves BBB function.
Fodor et al. aimed to find whether resveratrol could serve as a secondary prophylaxis agent for the prevention of stroke [229].They found that resveratrol supplements consumption for 12 months lowered the major risk factors associated with stroke including levels of total cholesterol, triglycerides, HDL and LDL cholesterol, basal glucose, and glycosylated hemoglobin (HbA1c).
These studies lacked measurement of long-term outcomes and to determine whether resveratrol supplement can prevent the occurrence of IS or at least decrease stroke severity and neurologic deficit degree.Future studies should consider these limitations.The outcomes of these trials are summarized in Table 3.

Conclusions
Inflammation and oxidative stress play a crucial role in stroke pathogenesis.The immune response followed by stroke triggers a cascade of events that lead to BBB dysfunction and the aggravation of neurologic deficits.Resveratrol effects multiple pathways, M. Owjfard et al. including SIRT1, Nrf2, and Shh which overall lead to reduced inflammation, oxidative stress, and better stroke outcomes.Resveratrol exhibits anti-inflammatory, anti-oxidative, anti-apoptotic and edema reducing effects in the stroke setting.Studies that have demonstrated resveratrol's neuroprotective role and its impact on improved stroke outcomes are mainly animal studies.Although the results of animal studies are highly generalizable to the clinical setting, future clinical trials are required to assess the potential of resveratrol in the long term and to determine whether pre-treatment with resveratrol could prevent or at least decrease stroke severity.Clinical trials have found that resveratrol reduces the factors associated with increased stroke risks such as lipid profile and blood glucose, which need further attention.Blood pressure management is a crucial matter in stroke, both in prevention and intervention.The effects of resveratrol on improving endothelial function and regulating BP seem to improve CBF.In the meantime, it seems that further studies are required to better clarify if resveratrol could serve as a therapeutic agent in the stroke setting.

Limitation
In the present study, we used all the articles in English, where the full text was available, and the subject under our investigation was based on the anti-inflammatory, anti-apoptotic, and antioxidant properties of resveratrol, including animal studies and clinical trials.

M
.Owjfard et al.

Table 1
Animal experiments with resveratrol in the brain injury.

Table 1 (
continued ) the WT MCAO group but did not have an effect in the Nrf2 − /− group (continued on next page) M.Owjfard et al.

Table 2
In vitro experiments with resveratrol in ischemic conditions.

Table 3
Clinical trials with resveratrol in ischemic stroke.